Improved detection of methylation in ancient DNA
Susanna SawyerPere GelabertBenjamin YakirAlejandro Llanos LizcanoAlessandra SperdutiLuca BondioliOlivia CheronetChristine Neugebauer-MareschMaria Teschler‐NicolaMario NovakIldikó PapIldikó SzikossyTamás HajduEran MeshorerLiran CarmelRon Pinhasi
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Abstract Reconstructing premortem DNA methylation levels in ancient DNA (aDNA) has led to breakthrough studies such as the prediction of anatomical features of the Denisovan, as well as the castration status of ancient horses. These studies relied on computationally inferring methylation levels from damage signals in naturally deaminated cytosines. Because of statistical constraints, this inference requires high-coverage sequencing, and is thus not only expensive but also restricted to samples with exceptional DNA preservation. Instead, a method to directly measure methylation levels in aDNA, as exists in modern DNA samples, would open the door to a more thorough and cost effective ability to study ancient DNA methylation. We have tested two methods for direct methylation measurement developed for modern DNA based on either bisulfite or enzymatic methylation treatments. We find that both methods preserve sufficient DNA yields to allow for methylation measurement. Bisulfite treatment, combined with a single stranded library preparation, shows the least reduction in DNA yields compared to no methylation treatment, as well as the least biases during methylation conversion. In addition, we show that applying bisulfite treatment to ∼0.4-fold coverage sample provides a methylation signal that is comparable to, or even better, than the computationally inferred one. We thus present a method to directly measure methylation in ancient DNA that is cost effective and can be used on a wide variety of ancient samples.Keywords:
Ancient DNA
Bisulfite
Bisulfite sequencing
Illumina Methylation Assay
Methylated DNA immunoprecipitation
CpG site
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Abstract Bisulfite sequencing has long been considered the gold standard for measurement of DNA methylation at single CpG resolution. In the meantime, several new approaches have been developed, which are regarded as less error-prone. Since these errors were shown to be sequence-specific, we aimed to verify the methylation data of a particular region of the TRPA1 promoter obtained from our previous studies. For this purpose, we compared methylation rates obtained via direct bisulfite sequencing and nanopore sequencing. Thus, we were able to confirm our previous findings to a large extent.
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DNA cytosine methylation is a central epigenetic modification that has essential roles in the control of many critically important biological processes,including cell proliferation,differentiation,development,genomic imprinting and regulation of gene expression.With progresses in study on DNA methylation,detection techniques of DNA methylation have been developed to meet requirements of various researches.This article has established an improved bisulfite sequencing technique for analysis of MutL-homologue 1(MLH1) promoter methylation level of mismatch repair gene in Arabidopsis thaliana.After the genomic DNA in Arabidopsis thaliana was digested,purified,and then modified with bisulfite liquid,the PCR products were cloned into the pEASY-T1 vector.15 positive clones were randomly selected from each sample and then sequenced.The results showed that compared with the traditional method of methylation analysis,the improved method modified the digested DNA completely,reduced the modification time greatly,and improved the specificity,stability and repeatability of the PCR products.MLH1 promoter methylation level was 27.3% instead of 45.6%.The improved bisulfite sequencing technique avoided misidentification at non-methylated sites,and would provide a better means of research for analysis of DNA methylation in plant.
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